The long-term objective of this project is to explore the unusual uptake mechanism utilized for lacrimal acinar cell internalization of adenovirus serotype 5, and to learn enough about this pathway to potentially use it for targeted drug delivery of therapeutic molecules to the lacrimal gland. More specifically, this project seeks to investigate the trafficking mechanisms of the coxsackievirus and adenovirus receptor (CAR) in rabbit lacrimal gland acinar cells (LGACs). The first specific aim is "How does CAR facilitate internalization of Ad5, fiber, or knob?" This aim seeks to investigate whether CAR is directly involved in binding and/or internalization of Ad5 into LGACs. The research approach that will be used to investigate CAR'S specific role in Ad5 uptake into LGACs is classical binding and internalization studies using radiolabeled virus, fiber, and knob performed with CAR-specific siRNA treated or untreated LGACs. The rationale for these studies is that transduction of LGAC by Ad5 is significantly reduced upon siRNA knockdown of CAR. However, it remains to be determined whether the effect is due to reduced binding and/or reduced internalization. If CAR knockdown is shown to affect internalization, we will investigate whether CAR itself is internalized in the presence of Ad5, fiber, or knob. The approach for these studies is analysis of the intracellular localization of an over-expressed, epitope-tagged CAR (HA-CAR) with and without Ad5, fiber, and knob exposure, by confocal fluorescence microscopy. If CAR is detected intracellularly, we will investigate its colocalization with a variety of fluorescent membrane compartment markers, including markers for early endosomes and lysosomes. This analysis will also be done by confocal fluorescence microscopy. The second specific aim is "Is CAR trafficked along with the viral capsid proteins?" If CAR is shown to be internalized, this aim seeks to investigate its intracellular trafficking pathways as well as its colocalization with viral capsid proteins. These studies also use the HA-CAR viral construct for colocalization studies with endosomal markers and viral capsid proteins using confocal fluorescence microscopy. In addition, chemical inhibitors of endocytic pathways will be used to confirm the endocytic pathway used by CAR. Dry eye diseases affect 10-14 million people in the U.S. alone. To date, the only successful gene delivery strategy (in animal models) is inadequate for clinical trials because of its potential to cause an immune response. A newer approach involves the use of viral capsid proteins to exploit specific and unique uptake mechanisms in the lacrimal gland for delivery of therapeutic molecules.